The invention concerns an illumination device for a scanning microscope. More specifically the invention concerns an illumination device for a scanning microscope for illuminating a specimens in a scanning microscope, which defines a laser beam. The invention furthermore concerns a method for illumination of specimens with such a illumination device. Additionally the invention concerns a scanning microscope.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light thereupon emitted by the specimen, laser beams usually being used for illumination. The specimen is scanned by means of a finely focused light beam. In confocal scanning microscopy specifically, the specimen is scanned in three dimensions with the focus of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture stop (called the xe2x80x9cexcitation stopxe2x80x9d), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels through the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection stop behind which the detectors (usually photomultipliers) are located. Detected light that does not derive directly from the focus region takes a different light beam path and does not pass through the detection stop, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image.
In many of these applications the wavelength of the illuminating light used, i.e. in particular the wavelength of the laser light that is utilized, is of critical importance. For the examination of cell-biology specimens, for example, the latter can be marked with different dyes in various regions. The specimens marked in this fashion can then be examined by means of confocal scanning microscopy, in which context the various dyes are excited to fluoresce. This requires, however, the application of a wavelength with which the particular dye can be excited. Since these wavelengths vary depending on the dye, in the so-called one-photon method it is necessary to perform the examination with the particular appropriate wavelengths.
In order to minimize stress on the samples during examination, the so-called two-photon method is already used. In this, a specimen labeled with dyes is irradiated using lasers that emit ultra-short laser pulses which at the same time have a high peak pulse power. At the focus of this laser radiation, the available photon density is high enough to produce nonlinear optical effects, for example two-photon absorption. Using this effect, it is possible to excite dyes with photons whose energy corresponds to only half the excitation energy inherently required, although two such photons participate in the production of this excitation state and are also necessary for it. The number of suitable lasers at the corresponding wavelengths is limited, however, so that the capabilities for examining the sample are limited to the use of a few dyes that match those wavelengths.
U.S. Pat. No. 6,154,310 has already proposed using a nonlinear optical effect in waveguides which makes it possible to modify the wavelength of a laser that generates ultra-short pulses. This is done by conveying the laser pulse to a plurality of waveguide elements. In each of these waveguide elements, the wavelength of the laser light is modified individually. The divided light beams from the plurality of waveguides are then combined back into a single beam. The result is to generate a new output wavelength that can be used for microscopy. Although the wavelength region can thereby be expanded beyond the usual effective range of a titanium/sapphire (TiSa) laser, the number of attainable wavelengths still remains limited.
Since it is desirable in scanning microscopy, inter alia, to use one-photon and two-photon excitation on a specimen under examination during a microscopy operation, it is nevertheless necessary to utilize different light sources, in particular different laser light sources, having different wavelengths, and to be able to switch between them. It is therefore usual to use for illumination of the specimen a number of radiation sources that are coupled into a microscope through so-called ports between which it is possible to switch. In particular, a mode-coupled TiSa laser can be coupled into the so-called two-photon port, and conventional gas lasers for UV and/or visual excitation can be coupled into the one-photon port. A switchover between the two ports is possible but very complex, since at least two acoustooptical tunable filters (AOTFs), acoustooptical modulators (AOMs), and/or electrooptical modulators (EOMs) are required, as well as a special control board with which the circuit can be activated and synchronized. Even expansion of the wavelength region of a TiSa laser as described above cannot satisfactorily solve this problem, since in this context the laser beam is split using a beam splitter, the first part being used for frequency conversion and the other part directly for illumination of the specimen. Thus only a portion of the energy is available for the two parts of the beam, so that on the one hand the illumination intensity on the sample is less than is desirable, and on the other hand the maximum laser energy is not available for frequency conversion. In addition, utilization of a beam splitter defines a fixed division ratio for the two partial beams. A different fixed division ratio is possible only by replacing the beam splitter. This switchover demands time and possibly alignment. Continuous or rapid changing between different division ratios is not possible.
The object of the present invention is accordingly to propose an illumination device for a scanning microscope which easily incorporates two or more illumination conditions.
According to the present invention, this object is achieved by an illumination device for a scanning microscope which comprises:
an illumination source for generating a laser beam;
a switchable beam deflection device which directs, in a first switching state, the laser beam along a first beam path, and in a second switching state, along an alternative beam path; and
a device for frequency conversion of the laser beam is arranged in the beam path of the alternative beam path.
It is a further object of the present invention to propose an illumination device for a scanning microscope which easily incorporates two or more illumination conditions.
According to the present invention, this object is achieved by an illumination device for a scanning microscope which comprises:
an illumination source for generating a laser beam;
a plurality of switchable beam deflection devices, wherein each of which directs, in a first switching state, the laser beam along a first beam path, and in a second switching state, along an alternative beam path and the plurality of switchable beam deflection devices and multiple alternative beam paths are provided in parallel with one another; and
a device for frequency conversion of the laser beam is arranged each beam path of the alternative beam path.
It is an object of the present invention to propose a scanning microscope which easily can perform two or more illumination conditions applied to a specimen.
The object is accomplished by a scanning microscope which comprises:
a two photon port and a one-photon port;
an illumination source for generating a laser beam;
at least one switchable beam deflection device which directs, in a first switching state, the laser beam along a first beam path to the two photon port, and in a second switching state, along an alternative beam path to the one-photon port; and
a device for frequency conversion of the laser beam is arranged in the beam path of the alternative beam path.
A further object of the present invention is to provide a method for the illumination of a specimen in a scanning microscope, which easily has two or more illumination conditions applied to a specimen.
The above object is accomplished by a method which comprises the steps of:
providing a laser which defines an illumination source for the specimen;
directing the laser beam is directed onto a switchable beam deflection device;
directing the laser beam from the switchable beam deflection device in substantially unattenuated fashion along a first beam path to the two photon port or in substantially unattenuated fashion along an alternative beam path to a one-photon port;
modifying the laser beam, prior to the one-photon port, along the alternative beam path with respect to its frequency; and
passing laser beam onto the specimen.
According to the present invention, a device for illumination of the specimens to be examined is provided in a scanning microscope, a laser radiation firstly being generated with a laser. This laser radiation is directed via an optical system onto the specimen to be examined. A switchable deflection device, with which it is possible to convey the incident laser beam either along a first beam path or along an alternative beam path to the microscope and thus to the specimen, is provided in the beam path of the illumination device. The switchable beam deflection device is preferably embodied in such a way that it directs the incident laser radiation substantially entirely along the first or substantially entirely along the alternative beam path. In very particularly preferred fashion, the beam deflection device is embodied in such a way that the division ratio of the incident laser radiation along the first and along the alternative beam path is continuously variable. While the laser radiation along the first beam path remains substantially unchanged at least in terms of its frequency, a component for frequency conversion of the laser beam is provided in the beam path of the alternative beam path. It is thus possible to use the first beam path for a first illumination type and the second beam path for a second illumination type. Illumination of the specimen under different illumination conditions can thus be achieved with a single illumination source (for example, a short-pulse laser). The switchable beam deflection device is preferably embodied in such a way that a rapid switchover between the first and the alternative beam path becomes possible. Optionally, a rapid switchover between different division ratios of the laser radiation along the first and along the alternative beam path is achievable. Usually, laser radiation along the first beam path is coupled into the so-called two-photon port, and laser radiation along the alternative beam path is coupled into the one-photon port, since one-photon excitation and two-photon excitation can be brought about in this fashion.
In a preferred embodiment, a mode-coupled TiSa short-pulse laser is used as the usable output light source. An electrooptical modulator (EOM) connected to a voltage source can be used as the rapidly switchable beam deflection device. The voltage source acts on the EOM in such a way that the polarization direction of the incident light can be rotated. If a polarization beam splitter is then provided in the beam path, the maximum laser power can thus either be used for illumination of the specimen along the first beam path via the two-photon port, or made available at full power for frequency conversion along the alternative beam path. The combination of an EOM (and in particular, associated therewith, the ability to set any desired polarization direction of the laser radiation) and a polarization beam splitter moreover makes possible any desired variation in the laser power along the first and the alternative beam path.
Any component with which it is possible to modify the frequency of an incident illuminating radiation is a possibility as the means for frequency conversion. In particular, the frequency can also be broadened during the frequency conversion, so that polychromatic light, from which the desired frequency band can be picked out by means of a suitable filter, is present at the output of the frequency conversion. With the device according to the present invention it is therefore possible, especially in the context of biological or chemical processes that are to be observed using a scanning microscope, to use two different forms of excitation and to switch rapidly between the two. With line-by-line scanning of the specimen, for example, it is also possible to switch over in line-by-line fashion between the one-photon and the two-photon port. High resolution in time can thereby be achieved, which is very important in particular for biological and chemical processes.
The switchover can, of course, also be accomplished in the context of so-called region-of-interest (ROI) scanning. In this, the specimen is illuminated in general with laser light from the one-photon port, and operation is switched over to the two-photon port in regions of particular interest, so that within the ROIs, scanning takes place with reduced specimen stress using the two-photon port.